This application addresses broad Challenge Area (04) Clinical Research and specific Challenge Topic, 04-AR-101: Autoimmunity For Diseases Of The Skin, Joints, Muscle And Other Tissues-Develop reagents and analytic methods to identify, characterize, track, and inhibit human B and T cells specific for defined selfantigens, and antigen-presenting cells. Autoimmune diseases such as multiple sclerosis (MS), type 1 diabetes (T1D) and rheumatoid arthritis (RA) are complex genetic diseases mediated by activated, autoreactive T cells and B cells. The presence of clonally expanded populations of T cells and B cells in the inflamed tissue of patients has long suggested that self-antigens drive the disease process. A major challenge hindering the study of human autoimmune diseases is the reliable identification and characterization of autoreactive B and T cells. These rare, self-reactive cells are present in the periphery, albeit at very low frequencies (>1 in 10,000). The technologies used for studying these cells include flow cytometry and immunosorbant assays, but these methods either have insufficient sensitivity for detecting low-frequency cells or the recovery of the cells is not possible. Significant advances in the understanding of the etiologies of autoimmune diseases would be possible if it were feasible to assess the frequency, clonal variations, and functional responses of autoreactive B and T cells in these diseases directly ex vivo. The central goal of this research is to establish a novel set of methods that use microfabricated systems to isolate and detect both self-reactive B and T cells. This project involves a collaboration amongst: the Wucherpfennig lab (Dana Farber) with expertise in recombinant antigens for MS to identify B and T cells, the Hafler lab (Brigham and Women's) with expertise in cloning and characterizing autoreactive T cells in MS, and the Love lab (MIT) with expertise in simultaneous parallel analyses of secreted products from >105 single primary cells. Specifically, we will develop single-cell multiparametric assays using arrays of nanowells to recover antigen-specific B cells from pediatric and adult MS patients. In a parallel aim, we will also develop a complementary method to identify autoreactive T cells in MS patients. The microstructure common to both of these aims-the arrays of nanowells-will enable an integrated technology platform capable of comprehensive characterization of both compartments of selfreactive lymphocytes. We will use these technologies to profile B cell responses in pediatric MS patients and evaluate the frequencies and clonal diversity in self-reactive T cells from MS patients. The outcome of these studies will be specific unprecedented knowledge on the breadth of B and T cell populations in MS that has not been accessible previously, and more broadly, a general process for evaluating these types of self-reactive cells in many human autoimmune diseases. PUBLIC HEALTH RELEVANCE: Autoimmune diseases are challenging to study because the cells involved in the disease-self-reactive B and T cells-are rare. Understanding the nature and diversity of these cells involved in the progression of diseases such as multiple sclerosis and type 1diabetes would aid in the development of new therapies, but the cells are difficult to detect and isolate with existing technologies. The aim of this project is to develop new technologies based on microfabricated systems that will allow the isolation and characterization of both B and T cells relevant for autoimmune diseases, but also for other human diseases.